Cathepsin D has been purified from porcine spleen to near homogeneity. Polyacrylamide electrophoresis of the purified enzyme showed one major band with only minor impurities. The purified enzyme has a m. wt. of 35,000. It contains 2 distinct optimal pH's when hemoglobin is the substrate. The formate and/or acetate apparently shifted the pH-dependence of the activity. The kinetic studies of cathepsin D have been made feasible by the isolation of a substrate which is a 32-residue CNBr-fragment of human hemoglobin. The continuation of this project is aimed at the studies of kinetics, partial structure and the nature of active center of this enzyme.